CN107058613A - The EBV DNA one-step method that a kind of sample is directly added into determines kit - Google Patents
The EBV DNA one-step method that a kind of sample is directly added into determines kit Download PDFInfo
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- CN107058613A CN107058613A CN201611195328.9A CN201611195328A CN107058613A CN 107058613 A CN107058613 A CN 107058613A CN 201611195328 A CN201611195328 A CN 201611195328A CN 107058613 A CN107058613 A CN 107058613A
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Abstract
Kit is determined the invention provides the EBV DNA one-step method that a kind of sample is directly added into, belongs to medical science molecular biosciences detection technique.Kit mainly includes the nucleic acid amplification agents (including EBV PCR reaction solutions and enzyme mixation) that (1) is prepackaged in PCR eight unions of amplification；(2) nucleic acid releasing agent；(3) quantitative calibration product and quality-control product (including critical positive quality control product, strong positive quality-control product and negative quality-control product).Detection reagent is dispensed into PCR and expanded in eight unions by the present invention in advance, and nucleic acid releasing agent is added when using and testing sample carries out real-time fluorescence quantitative PCR.The detectable Epstein-Barr virus carrying capacity included but is not limited in a variety of samples such as serum, blood plasma and urine of the present invention, and specific add internal standard in EBV PCR reaction solutions, it is ensured that testing result it is authentic and valid.The present invention it is simple to operate, repeatability and have good stability, be adapted to use in each stratum's medical research unit.
Description
Technical field
The present invention relates to biology field, the EBV DNA one-step method that more particularly to a kind of sample is directly added into is determined
Kit.
Background technology
Epstein-Barr virus (Epstein-Barr virus, EBV) is a kind of common nerpes vinrus hominis, is that infectiousness monokaryon is thin
The main pathogen of born of the same parents' increase disease, it is relevant with a variety of human diseases.Crowd of the whole world about more than 90% infected EBV.EBV can tire out
Count the multiple systems of whole body, including liver, spleen, lymph node, kidney, heart, lung, marrow and brain etc., and clinical shape variation.EBV sense
Dye rate is high, and the generation development with a variety of diseases is inseparable, common disease have hepatitis, ITP,
Organ transplant postoperative infection, HSCT postoperative infection etc..Therefore, examination and auxiliary of the detection EBV content to disease is examined
It is disconnected significant.
PCR (Polymerase Chain Reaction, PCR) is a kind of multiple according to DNA in organism
Property processed and the technology of external rapid amplifying specific dna sequence designed.PCR reaction systems it is main by nucleic acid primer, 4 kinds
DNTPs, archaeal dna polymerase, template DNA and PCR reaction buffers system composition.From U.S. Cetus companies human inheritance room Kary
Mullis and its work together since 1985 invention PCR (PCR), round pcr and its deriving technology are just quick
Develop, and extensive utilization has been obtained in multiple nucleic acids detection.The especially detection of virus or other pathogens, when knowing
During a certain specific gene fragment of road cause of disease to be checked, you can enter performing PCR using target dna micro in specific primer pair sample
Amplification, reaches detection limit, is detected by appropriate detection means, you can determine the presence or absence of pathogen.
Real-Time Fluorescent Quantitative PCR Technique, refers to add fluorophor in PCR reaction systems, during DNA cloning
The accumulation of fluorescence signal monitors whole PCR processes in real time, carries out the side of quantitative analysis to unknown template finally by standard curve
Method.Compared with common PCR.Real-Time Fluorescent Quantitative PCR Technique has high specificity, fast spends high, reproducible, quantitative standard
Really, speed is fast, totally-enclosed reaction the advantages of, realize PCR quantitative analysis.At present, Real-Time Fluorescent Quantitative PCR Technique is in doctor
It is widely applied in, agriculture and animal husbandry, the quantitative study of bio-related molecules biology and clinical detection.
The content of the invention
Reagent is determined the technical problem to be solved in the present invention is to provide the EBV DNA one-step method that a kind of sample is directly added into
Box, while providing corresponding kit based on this method.
In order to solve the above problems, the scheme that the present invention is provided is：
The EBV DNA one-step method that a kind of sample is directly added into determines kit, it is characterised in that kit mainly includes (1)
It is prepackaged in the nucleic acid amplification agents (including EBV-PCR reaction solutions and enzyme mixation) in PCR eight unions of amplification；(2) nucleic acid
Releasing agent；(3) quantitative calibration product and quality-control product (including critical positive quality control product, strong positive quality-control product and negative quality-control product).
In the present invention, inventor is in advance by nucleic acid amplification agents (mainly including EBV-PCR reaction solutions and enzyme mixation)
It is dispensed into PCR to expand in eight unions, and is closed with fusing point less than 42 DEG C in the paraffin oil of curdled appearance.Nucleic acid is released when in use
Put agent to be added in above the paraffin oil of solidification, fully reacted with the detection sample of addition, calibration object, quality-control product simultaneously.
Described EBV-PCR reaction solution includes target primer and probe, sequence in EBV primer and probe and specificity
Row are as follows：
EBV-F：5’-CCCGTCACGGTGACGTAGTCTGTCT-3’(SEQ ID NO：1)
EBV-R：5’-CTAGCAAAACCTCTAGGGCA-3’(SEQ ID NO：2)
EBV-probe：5’-HEX-TAGACACTGCAAAACCTCAGGACCTAC-BHQ1-3’(SEQ ID NO：3)
IC-F：5’-CAGCGAAGGCCGAAA AGA-3’(SEQ ID NO：4)
IC-R：5’-CATCCA ACGAACTCACCACTG T-3’(SEQ ID NO：5)
IC-probe：5’-FAM-AGCCATGCCCTTAGTAG-BHQ2-3’(SEQ ID NO：6)
The DNA sequence dna gone out by the primer and probe amplification of above-mentioned Epstein-Barr virus is：
CCCGTCACGGTGACGTAGTCTGTCTTGAGGAGATGTAGACTTGTAGACACTGCAAAACCTCAGGACCTACGCTGCCC
TAGAGGTTTTGCTAG(SEQ ID NO：7)；The DNA sequence dna gone out by the primer and probe amplification of above-mentioned internal standard (IC) is：
CAGCGAAGGCCGAAAAGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAATTGAGGGGGGTAGCAACAGTGGTGAGTC
GTTGGATG(SEQ ID NO：8).Wherein, EBV and internal standard IC 5 ' ends marked different fluorescence probe report groups, wherein
EBV fluorescence probe report group is HEX, and quenching group is BHQ1；The fluorescence probe report group of internal standard (IC) is FAM, sudden
Group go out for BHQ2.
Preferably, EBV-PCR reaction solutions system includes primer, probe, Tris alkali, magnesium chloride, potassium chloride and dNTP.
Preferably, the composition of nucleic acid releasing agent includes：0.2~0.4N sodium hydroxides, 0.3~0.6M potassium chloride, 0.01
One kind in~0.05%N- sodium lauroyl sarcosines, 5mM EDTA, 0.3~0.6M Tris-HCL and 1~2% triton x-100
Or it is a variety of.
Preferably, described nucleic acid releasing agent is added in above solidification paraffin oil, with the detection sample of addition simultaneously, calibration
Product, quality-control product fully react.
Preferably, the method for the invention comprises the following steps：
Step 1) PCR for packaging nucleic acid amplification agents in advance is expanded and managed, xeothermic 43 DEG C of device rewarming 1-2 minutes is placed in,
It is placed on room temperature to cool down 1 minute, solidifies saxol.
Step 2) remove PCR amplification pipe on cover layer, be directly added into 10 μ l nucleic acid releasing agents and 20 μ l testing samples, cover
Eight union lids, reverse mixing 3-5 times, avoid light place 5-10 minutes.
Step 3) PCR amplification pipes are placed in xeothermic 72 DEG C of device rewarming 1-2 minutes, it is inverted under ttom of pipe liquid gently bullet, makes
Ttom of pipe nucleic acid releasing agent and sample are thoroughly mixed with bottom PCR reaction solutions,
Step 4) moment centrifugation, directly carry out quantitative fluorescent PCR.Wherein quantitative fluorescent PCR response procedures are 37 DEG C,
2min；95 DEG C, 15 minutes；Then 95 DEG C, 15 seconds and 60 DEG C, 45 seconds of 45~50 circulations are carried out；Fluorescence is detected at 60 DEG C
Signal.
Step 5) interpretation of result.According to the Ct values of standard items supporting in kit, the virus quantity in testing sample is entered
The accurate definite value of row.
Preferably, the testing sample includes but is not limited to serum, blood plasma or urine etc., preferably serum or blood plasma.
Beneficial effects of the present invention are：
The present invention in order to realize efficiently, efficiently detection sample in EBV viral levels, according to EBV virus genome sequence
Row, devise the viral specific primer and probe.Meanwhile, whether proper in order to monitor experimentation, the present invention is also included
Non- plasmid-type internal standard, the internal standard is selected from non-human's virus sequence, and sample will not be polluted, and can stablize be expressed in it is whole
Individual experimentation.In order to effectively shorten experiment process, reduce the pollution that external environment is caused to experiment, the invention general
Nucleic acid amplification agents are dispensed into PCR and expanded in eight unions in advance, and the paraffin oil with fusing point less than 42 DEG C is enclosed in ttom of pipe.
Nucleic acid releasing agent and testing sample need to be only added when in use.The experiment proved that, this method can not only shorten test into
Journey, and accurate quantitative analysis can be carried out to the EBV viruses in sample.Specific primer can expand the standard of testing sample or configuration simultaneously
Product, viral level that can precisely in special response sample, and do not disturb mutually.
Kit is determined the invention provides the EBV DNA one-step method that a kind of sample is directly added into.Preferably, of the invention
Kit includes the standard items of EBV viruses, available for drafting standard curve, and the viral level in sample is carried out accurately to determine
Amount.
Brief description of the drawings
Fig. 1 detects EBV DNA result figure for the method for the embodiment of the present invention 1；
Fig. 2 detects EBV DNA result figure for the method for the embodiment of the present invention 2；
Fig. 3 is the susceptibility results figure that EBV DNA are detected using the inventive method；
Fig. 4 is the repeated result figure that EBV DNA are detected using the inventive method；
Embodiment
Kit, those skilled in the art are determined the invention discloses the EBV DNA one-step method that a kind of sample is directly added into
Present disclosure can be used for reference, technological parameter realization is suitably modified.It is important to note that all similar replacements and change pair
It is that it will be apparent that they are considered as being included in the present invention, and related personnel substantially can be for those skilled in the art
Do not depart from and content described herein be modified on the basis of present invention, spirit and scope or suitably change is with combining,
Realize and apply the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The implication that personnel are generally understood that.Meanwhile, for a better understanding of the present invention, the definition and explanation of relational language is provided below.
The term " nucleic acid releasing agent " used in the present invention also known as " lysate ", i.e., " lysis buffer ", refer in order to
The nucleic acid in sample is set to be free in a kind of preparation liquid added needed for cracking system.
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.
Experiment material and instrument
PCR amplification instrument device used is the real-time fluorescence that Shanghai Hong Shi medical science and technologies Co., Ltd produces in following examples
Quantitative PCR apparatus.Sample to be tested uses the EBV DNA blood serum samples determined through clinical assay and EBV standard items to be detected.
Embodiment 1：EBV real-time fluorescence quantitative PCRs are detected
By clinical diagnosis clearly and by detecting that EBV DNA quantitative values are 1 × 102IU/ml patients serum's sample according to
Following steps carry out real-time fluorescence quantitative PCR detection.
(1) design of Epstein-Barr virus and internal standard specific primer and probe
For detecting that EBV and internal standard (IC) primer and probe sequence are as follows：
EBV-F：5’-CCCGTCACGGTGACGTAGTCTGTCT-3’(SEQ ID NO：1)
EBV-R：5’-CTAGCAAAACCTCTAGGGCA-3’(SEQ ID NO：2)
EBV-probe：5’-HEX-TAGACACTGCAAAACCTCAGGACCTAC-BHQ1-3’(SEQ ID NO：3)
IC-F：5’-CAGCGAAGGCCGAAA AGA-3’(SEQ ID NO：4)
IC-R：5’-CATCCA ACGAACTCACCACTG T-3’(SEQ ID NO：5)
IC-probe：5’-FAM-AGCCATGCCCTTAGTAG-BHQ2-3’(SEQ ID NO：6)
The DNA sequence dna gone out by the primer and probe amplification of above-mentioned Epstein-Barr virus is：
CCCGTCACGGTGACGTAGTCTGTCTTGAGGAGATGTAGACTTGTAGACACTGCAAAACCTCAGGACCTACGCTGCCC
TAGAGGTTTTGCTAG(SEQ ID NO：7)；The DNA sequence dna gone out by the primer and probe amplification of above-mentioned internal standard (IC) is：
CAGCGAAGGCCGAAAAGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAATTGAGGGGGGTAGCAACAGTGGTGAGTC
GTTGGATG(SEQ ID NO：8).Wherein, EBV and internal standard IC 5 ' ends marked different fluorescence probe report groups, wherein
EBV fluorescence probe report group is HEX, and quenching group is BHQ1；The fluorescence probe report group of internal standard (IC) is FAM, sudden
Group go out for BHQ2.
(2) extraction of viral nucleic acid and real-time fluorescence quantitative PCR
Step 1) PCR for packaging nucleic acid amplification agents in advance is expanded and managed, xeothermic 43 DEG C of device rewarming 1-2 minutes is placed in,
It is placed on room temperature to cool down 1 minute, solidifies saxol.
Step 2) remove PCR amplification pipe on cover layer, be directly added into 10 μ l nucleic acid releasing agents and 20 μ l testing samples, cover
Eight union lids, reverse mixing 3-5 times, avoid light place 5-10 minutes.
Step 3) PCR amplification pipes are placed in xeothermic 72 DEG C of device rewarming 1-2 minutes, it is inverted under ttom of pipe liquid gently bullet, makes
Ttom of pipe nucleic acid releasing agent and sample are thoroughly mixed with bottom PCR reaction solutions,
Step 4) moment centrifugation, directly carry out quantitative fluorescent PCR.Wherein quantitative fluorescent PCR response procedures are 37 DEG C,
2min；95 DEG C, 15 minutes；Then 95 DEG C, 15 seconds and 60 DEG C, 45 seconds of 45~50 circulations are carried out；Fluorescence is detected at 60 DEG C
Signal.
Step 5) interpretation of result.According to the Ct values of standard items supporting in kit, the virus quantity in testing sample is entered
The accurate definite value of row.
Wherein, the EBV-PCR reaction solutions concrete composition composition is as follows：PCR Master Mix 20ul, 5uM EBV-F
0.5ul, 5uM EBV-R 0.5ul, 5uM EBV-probe 0.5ul, 5uM IC-F 0.5ul, 5uM IC-R 0.5ul, 5uM
IC-probe 0.5ul and ddH2O 5.5ul.
Wherein, the PCR Master Mix constituents include：500mM Tris-HCl(pH8.0)、500mM KCl、
15mM MgCl2 and 10mM dNTPs.
Wherein, the composition of the nucleic acid releasing agent includes：0.2N sodium hydroxides, 0.3M potassium chloride, 5mM EDTA, 0.01%
One or more in N- sodium lauroyl sarcosines, 0.3Tris-HCL and 1% triton x-100.
(3) interpretation of result
Experimental result according to the detection method of this example, effectively can accurately detect EBV as shown in figure 1, as a result show
DNA quantitative values are 1 × 102IU/ml EBV patients serum's samples.
Embodiment 2：EBV real-time fluorescence quantitative PCRs are detected
By clinical diagnosis clearly and by detecting that EBV DNA quantitative values are 1 × 102IU/ml patients serum's sample according to
Following steps carry out real-time fluorescence quantitative PCR detection.
(1) design of Epstein-Barr virus and internal standard specific primer and probe
For detecting that EBV and internal standard (IC) primer and probe sequence are as follows：
EBV-F：5’-CCCGTCACGGTGACGTAGTCTGTCT-3’(SEQ ID NO：1)
EBV-R：5’-CTAGCAAAACCTCTAGGGCA-3’(SEQ ID NO：2)
EBV-probe：5’-HEX-TAGACACTGCAAAACCTCAGGACCTAC-BHQ1-3’(SEQ ID NO：3)
IC-F：5’-CAGCGAAGGCCGAAA AGA-3’(SEQ ID NO：4)
IC-R：5’-CATCCA ACGAACTCACCACTG T-3’(SEQ ID NO：5)
IC-probe：5’-FAM-AGCCATGCCCTTAGTAG-BHQ2-3’(SEQ ID NO：6)
The DNA sequence dna gone out by the primer and probe amplification of above-mentioned Epstein-Barr virus is：
CCCGTCACGGTGACGTAGTCTGTCTTGAGGAGATGTAGACTTGTAGACACTGCAAAACCTCAGGACCTACGCTGCCC
TAGAGGTTTTGCTAG(SEQ ID NO：7)；The DNA sequence dna gone out by the primer and probe amplification of above-mentioned internal standard (IC) is：
CAGCGAAGGCCGAAAAGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAATTGAGGGGGGTAGCAACAGTGGTGAGTC
GTTGGATG(SEQ ID NO：8).Wherein, EBV and internal standard IC 5 ' ends marked different fluorescence probe report groups, wherein
EBV fluorescence probe report group is HEX, and quenching group is BHQ1；The fluorescence probe report group of internal standard (IC) is FAM, sudden
Group go out for BHQ2.
(2) extraction of viral nucleic acid and real-time fluorescence quantitative PCR
Step 1) PCR for packaging nucleic acid amplification agents in advance is expanded and managed, xeothermic 43 DEG C of device rewarming 1-2 minutes is placed in,
It is placed on room temperature to cool down 1 minute, solidifies saxol.
Step 2) remove PCR amplification pipe on cover layer, be directly added into 10 μ l nucleic acid releasing agents and 20 μ l testing samples, cover
Eight union lids, reverse mixing 3-5 times, avoid light place 5-10 minutes.
Step 3) PCR amplification pipes are placed in xeothermic 72 DEG C of device rewarming 1-2 minutes, it is inverted under ttom of pipe liquid gently bullet, makes
Ttom of pipe nucleic acid releasing agent and sample are thoroughly mixed with bottom PCR reaction solutions,
Step 4) moment centrifugation, directly carry out quantitative fluorescent PCR.Wherein quantitative fluorescent PCR response procedures are 37 DEG C,
2min；95 DEG C, 15 minutes；Then 95 DEG C, 15 seconds and 60 DEG C, 45 seconds of 45~50 circulations are carried out；Fluorescence is detected at 60 DEG C
Signal.
Step 5) interpretation of result.According to the Ct values of standard items supporting in kit, the virus quantity in testing sample is entered
The accurate definite value of row.
Wherein, the EBV-PCR reaction solutions concrete composition composition is as follows：PCR Master Mix 20ul, 5uM EBV-F
0.5ul, 5uM EBV-R 0.5ul, 5uM EBV-probe 0.5ul, 5uM IC-F 0.5ul, 5uM IC-R 0.5ul, 5uM
IC-probe 0.5ul and ddH2O 5.5ul.
Wherein, the PCR Master Mix constituents include：500mM Tris-HCl(pH8.0)、500mM KCl、
15mM MgCl2 and 10mM dNTPs.
Wherein, the composition of the nucleic acid releasing agent includes：0.4N sodium hydroxides, 0.6M potassium chloride, 5mM EDTA, 0.05%
One or more in N- sodium lauroyl sarcosines, 0.6Tris-HCL and 2% triton x-100.
(3) interpretation of result
Experimental result according to the detection method of this example, effectively can accurately detect EBV as shown in Fig. 2 as a result show
DNA quantitative values are 1 × 102IU/ml EBV patients serum's samples.
Embodiment 3：The sensitiveness and internal standard stability test of real-time fluorescence quantitative PCR detection of the present invention
With the serum sample that EBV DNA are feminine gender as dilution, by clinical diagnosis clearly and by detecting that EBV DNA determine
Value is 1 × 106IU/ml clinical samples are diluted, and it is 50IU/ml, 1 × 10 to obtain EBV DNA concentrations2IU/ml、1×
103IU/ml、1×104IU/ml、1×105IU/ml、1×106IU/ml sample uses it for sensitivity testses.Using with reality
Apply the identical method of example 1 and real-time fluorescence quantitative PCR detection is carried out to above-mentioned sample.
Experimental result is as shown in Figure 3：It is 50IU/ml, 1 × 10 that real linearity curve from right to left represents concentration according to this2IU/
ml、1×103IU/ml、1×104IU/ml、1×I05IU/ml、1×106IU/ml sample.As a result show, the present invention is detectable
Go out concentration range in 50IU/ml~1 × 106IU/ml EBV DNA samples, while showing that the sensitivity of the inventive method can be low
To 50IU/ml.Empty linearity curve is internal standard, and the interior target Ct values from as little as high various EBV virus concentrations are in 33 or so, explanation
The stable expression of internal standard is not influenceed by virus concentration.
Embodiment 4：The reperformance test and coefficient of variation analysis of real-time fluorescence quantitative PCR detection of the present invention
With the serum sample that EBV DNA are feminine gender as dilution, by clinical diagnosis clearly and by detecting that EBV DNA determine
Value is 1 × 106IU/ml EBV patients serums are diluted, and it is 50IU/ml, 1 × 10 to obtain EBV DNA concentrations2IU/ml、1
×103IU/ml、1×104IU/ml、1×I05IU/ml、1×106IU/ml sample.Use it for reperformance test.Using with
The identical method of embodiment 1 carries out 3 multiple holes detections to above-mentioned sample respectively.Observe the repeatability and the coefficient of variation of detection.Simultaneously
Sample is detected with the EBV detection commercially available reagent box sold on the market, the repeatability and the coefficient of variation of two methods is compared.
As a result as shown in Figure 4 and Table 1, it is 50IU/ml, 1 × 10 to concentration using the present invention that Fig. 4, which is,2IU/ml、1×
103IU/ml、1×104IU/ml、1×105IU/ml、1×106IU/ml sample carries out iterative testing, as shown in figure 4, its
Experimental result repeatability is good, and each concentration curve is basic at same Ct values.In addition, the internal standard curve corresponding to each concentration, such as
Also express stable shown in Fig. 4 dotted lines, Ct values are 33 or so.Table 1 is by the present invention and the EBV detection kits pair sold on the market
1×106IU/ml、1×104IU/ml、1×102Each concentration samples of IU/ml carry out 3 multiple holes and repeat to detect, observe Ct values and calculate
CV (%).As a result show, each concentration C t values of present invention detection are superior to each concentration C t for the EBV detection kits sold on the market
Value, 1 × 106IU/ml、1×104The coefficient of variation of two concentration of IU/ml and the EBV detection kits sold on the market are without system
Meter learns difference, and the present invention is 1 × 102The degree of variation of IU/ml concentration is significantly better than the EBV detection kits sold on the market
Degree of variation.So as to draw a conclusion：The detection repeatability and the coefficient of variation of the inventive method are better than the EBV detection examinations sold on the market
Agent box.
The reperformance test of the EBV DNA real-time fluorescence quantitative PCRs of table 1 detection and coefficient of variation analysis result
The above-described EBV detection kits sold on the market are applied to urine, serum sample.Its operating procedure is such as
Under：
(1) 100ul serum samples are taken, isometric concentrate, 12000rpm centrifugations 5min is added.
(2) supernatant is abandoned, 50ul nucleic acid releasing agents are added into precipitation.
(3) precipitation suction is provoked with liquid-transfering gun and plays mixing.
(4) sample to be tested after above-mentioned processing is added in each PCR reaction tubes.
(5) stand after 10min, often pipe adds PCR reaction solution 40ul, mixing bonnet upper tube cap and 2000rpm centrifugations are played in suction
30s。
(6) PCR is expanded.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. the EBV DNA one-step method that a kind of sample is directly added into determines kit, it is characterised in that it is pre- that kit mainly includes (1)
First it is packaged in the nucleic acid amplification agents (including EBV-PCR reaction solutions and enzyme mixation) in PCR eight unions of amplification；(2) nucleic acid is released
Put agent；(3) quantitative calibration product and quality-control product (including critical positive quality control product, strong positive quality-control product and negative quality-control product).
2. kit according to claim 1, it is characterised in that nucleic acid amplification agents (are mainly included into EBV-PCR reaction solutions
And enzyme mixation) be dispensed into advance in PCR eight unions of amplification, nucleic acid releasing agent is added when using and testing sample progress is glimmering in real time
Fluorescent Quantitative PCR.
3. kit according to claim 1 or claim 2, it is characterised in that described EBV-PCR reaction solutions include EBV primer
With target primer and probe in probe and specificity, sequence is as follows：
EBV-F：5’-CCCGTCACGGTGACGTAGTCTGTCT-3’(SEQ ID NO：1)
EBV-R：5’-CTAGCAAAACCTCTAGGGCA-3’(SEQ ID NO：2)
EBV-probe：5’-HEX-TAGACACTGCAAAACCTCAGGACCTAC-BHQ1-3’(SEQ ID NO：3)
IC-F：5’-CAGCGAAGGCCGAAAAGA-3’(SEQ ID NO：4)
IC-R：5’-CATCCA ACGAACTCACCACTG T-3’(SEQ ID NO：5)
IC-probe：5’-FAM-AGCCATGCCCTTAGTAG-BHQ2-3’(SEQ ID NO：6)
The DNA sequence dna gone out by the primer and probe amplification of above-mentioned Epstein-Barr virus is：
CCCGTCACGGTGACGTAGTCTGTCTTGAGGAGATGTAGACTTGTAGACACTGCAAAACCTCAGGACCTACGCTGCCC
TAGAGGTTTTGCTAG(SEQ ID NO：7)；The DNA sequence dna gone out by the primer and probe amplification of above-mentioned internal standard (IC) is：
CAGCGAAGGCCGAAAAGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAATTGAGGGGGGTAGCAACAGTGGTGAGTC
GTTGGATG(SEQ ID NO：8).Wherein, EBV and internal standard IC 5 ' ends marked different fluorescence probe report groups, wherein
EBV fluorescence probe report group is HEX, and quenching group is BHQ1；The fluorescence probe report group of internal standard (IC) is FAM, sudden
Group go out for BHQ2.
4. kit according to claim 1, it is characterised in that described EBV-PCR reaction solutions include primer, probe, Tris
Alkali, magnesium chloride, potassium chloride and dNTP.
5. according to claim 1,3 or 4, it is characterised in that described amplifing reagent (including EBV-PCR reaction solutions and enzyme it is mixed
Close liquid) it is coated in advance in PCR ' amplification pipes, closed using the paraffin oil less than 42 DEG C in curdled appearance.
6. it is according to claim 1 or claim 2, it is characterised in that the composition of described nucleic acid releasing agent includes：0.2~0.4N hydrogen-oxygens
Change sodium, 0.3~0.6M potassium chloride, 5mM EDTA, 0.01~0.05%N- sodium lauroyl sarcosines, 0.3~0.6M Tris-HCL
With the one or more in 1~2% triton x-100.
7. the kit according to claim 1,2 or 6, it is characterised in that described nucleic acid releasing agent is added on solidification paraffin oil
Side, fully reacts with the detection sample of addition, calibration object, quality-control product simultaneously.
8. the EBV DNA one-step method that a kind of sample is directly added into determines kit, it is characterised in that any using claim 1-7
Described detection kit is carried out, and specifically includes following steps：
Step 1) PCR for packaging nucleic acid amplification agents in advance is expanded and managed, xeothermic 43 DEG C of device rewarming 1-2 minutes is placed in, is placed
Cooled down 1 minute in room temperature, solidify saxol.
Step 2) remove PCR amplification pipe on cover layer, be directly added into 10 μ l nucleic acid releasing agents and 20 μ l testing samples, cover eight
Union lid, reverse mixing 3-5 times, avoid light place 5-10 minutes.
Step 3) PCR amplification pipes are placed in xeothermic 72 DEG C of device rewarming 1-2 minutes, it is inverted under ttom of pipe liquid gently bullet, makes ttom of pipe
Nucleic acid releasing agent and sample are thoroughly mixed with bottom PCR reaction solutions,
Step 4) moment centrifugation, directly carry out quantitative fluorescent PCR.Wherein quantitative fluorescent PCR response procedures are 37 DEG C, 2min；95
DEG C, 15 minutes；Then 95 DEG C, 15 seconds and 60 DEG C, 45 seconds of 45~50 circulations are carried out；Fluorescence signal is detected at 60 DEG C.
Step 5) interpretation of result.According to the Ct values of standard items supporting in kit, the virus quantity in testing sample is carried out accurate
It is determined that value.
9. according to claim 1, it is characterised in that the detected sample includes but is not limited to serum, blood plasma or urine
Deng preferably serum or urine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858451A (en) * | 2017-11-01 | 2018-03-30 | 中山大学肿瘤防治中心 | A kind of EBV capture probes and the method for obtaining EBV Genomic sequence informations in sample |
| CN108754024A (en) * | 2018-06-19 | 2018-11-06 | 长沙金域医学检验所有限公司 | A kind of quantitative detecting method of epstein barr virus dna |
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|---|---|---|---|---|
| CN103060473A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Herpes virus EBV (Epstein-Barr Virus) detection kit |
| CN104818338A (en) * | 2015-05-15 | 2015-08-05 | 王海滨 | Direct real-time fluorescent quantitative PCR method |
| CN105420403A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube |
-
2016
- 2016-12-22 CN CN201611195328.9A patent/CN107058613A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060473A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Herpes virus EBV (Epstein-Barr Virus) detection kit |
| CN104818338A (en) * | 2015-05-15 | 2015-08-05 | 王海滨 | Direct real-time fluorescent quantitative PCR method |
| CN105420403A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858451A (en) * | 2017-11-01 | 2018-03-30 | 中山大学肿瘤防治中心 | A kind of EBV capture probes and the method for obtaining EBV Genomic sequence informations in sample |
| CN108754024A (en) * | 2018-06-19 | 2018-11-06 | 长沙金域医学检验所有限公司 | A kind of quantitative detecting method of epstein barr virus dna |
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